Name: metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate
Uploaded: 2013-09-18T11:00:00
Description: Watch this Scientific Journal Video about Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate at JoVE.com

We are also grateful to the Michael J. Fox Foundation supporting this study. We thank the Research Foundation - Flanders FWO (FWO project G.0666.09, senior researcher fellowship to JMT), the IWT SBO/80020 project Neuro-TARGET, the KU Leuven (OT/08/052A and IOF-KP/07/ 001) for their support. This research was also supported in part by the Fund Druw&eacute;-Eerdekens managed by the King Baudouin Foundation to JMT.

The present protocol uses radioactive 32P-labeled orthophosphate to follow cellular phosphorylation of LRRK2. It is important to bear in mind that all operations with radioactive reagents should be performed using appropriate protective measures to minimize exposure of radioactive radiation to the operator and the environment. Compounds containing isotopes that emit ionizing radiation can be harmful to human health and strict licensing and regulations at an institutional and national level control their use. T...

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This paper provides a basic protocol for assaying general phosphorylation level of LRRK1 and LRRK2 in cell lines using metabolic labeling with 32P-orthophosphate. The overall strategy is straightforward. Affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively lab...

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain paralogs which share a similar domain organization. Both proteins encode a GTPase sequence akin to the Ras family of GTPases (Ras of Complex Proteins, or ROC) as well as a C-terminal of ROC domain (COR), effectively classifying both proteins to the ROCO protein family5,6. N-terminal of the ROC-COR domain tandem, both proteins encode a leucine-rich repeat domain as well as an ankyrin-like domain, while only LRRK2 encodes an extra armadillo domein6-8. C-terminal of ROC-COR, both proteins share a serine-threonine kinase domain while only LRRK2 encodes a WD40 domain in the C-terminal region8. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2 , while genetic evidence points to a role for LRRK2 in the pathogenesis of Parkinson's disease3,4 .
The phosphorylation of proteins is a common regulatory mechanism in cells. For example, phosphorylation can be essential for the activation of enzymes or for the recruitment of proteins to a signaling complex. The cellular phosphorylation of LRRK2 has been extensively characterized and phosphosite mapping has shown a majority of cellular phosphorylation sites to occur in a cluster between the ankyrin repeat and leucine rich repeat domains9-11. Although LRRK1 cellular phosphorylation sites have yet to be mapped, evidence from studies using phosphoprotein staining of blots of immunoprecipitated LRRK1 protein from COS7 cells suggests that LRRK1 protein is phosphorylated in cells12.
This paper provides a basic protocol for assaying general phosphorylation level of LRRK1 and LRRK2 in cell lines using metabolic labeling with 32P-orthophosphate. The overall strategy is straightforward. Affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. The affinity tag (3xflag) is then used to isolate the LRRK proteins from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Phosphorus-32 Radionuclide, 1 mCi, buffer disodiumphosphate in 1 ml water</td>
 <td>Perkin Elmer</td>
 <td>NEX011001MC</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose)</td>
 <td>Invitrogen</td>
 <td>11971-025</td>
 <td>This medium contains no phosphates</td>
 </tr>
 <tr>
 <td>Anti Flag M2 affiinty gel</td>
 <td>Sigma</td>
 <td>A2220</td>
 <td>For an equivalent product with red colored gel (useful to more easily visualize the beads), use cat. No. F2426.</td>
 </tr>
 <tr>
 <td>Extra thick blotting filter</td>
 <td>Bio-Rad</td>
 <td>1703965</td>
 <td></td>
 </tr>
 <tr>
 <td>Ponceau S solution</td>
 <td>Sigma</td>
 <td>P7170</td>
 <td></td>
 </tr>
 </tbody>
</table>

metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

In order to compare overall phosphorylation levels of LRRK1 and LRRK2 in cells, 3xflag tagged LRRK1 and LRRK2 were expressed in HEK293T cells15. Cells were cultured in 6-well plates and labeled with 32P and analyzed as described above in the protocol text. Figure 1 shows representative results for metabolic labeling of LRRK1 and LRRK2 in HEK293T cells. Radioactive phosphate incorporation is observed for both LRRK1 and LRRK2. Upon quantification of the 32P levels normalize...

Watch this Scientific Journal Video about Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate at JoVE.com

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

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